HLA-A位点PCR-SSP基因分型和血清学分型的比较

目的：建立优化ＨＬＡ－Ａ位点ＰＣＲ－ＳＰ分型方法。方法：采用普通扩增仪和Ｅｐｐｅｎｄｏｒｆ管对细胞系ＤＮＡ和３７个健康正常人进行基因分型,血清学检测采用标准淋巴细胞毒试验方法。结果：发现引物浓度和浓度比、ＤＮＡ的纯度及酶的选用,是影响ＨＬＡ－Ａ位点ＰＣＲ－ＳＳＰ分型准确性的重要因素。该方法对３７个标本的基因分型结果与血清学结果相符合。结论：ＰＣＲ－ＳＳＰ方法具有简便准确的优点,可以作为ＨＬＡ－Ａ位     

用PCR—SSP作HAL—B位点分型及B22亚型分析

目的：用ＤＮＡ分型弥补血清学ＨＬＡ－Ｂ位点分型的欠缺并了解南方汉族Ｂ２２亚型分布情况。方法；采用标准细胞作参照，建立Ｂ位点的ＰＣＲ－ＳＳＰ分型方法地血清学Ｂ２２阳性标本进行Ｂ２２亚型分析。结果：１０４株标准细胞ＰＣＲ－ＳＳＰ分型结果与已知结果完全一致，１７例白血病人及同胞分型与血清学一致，１例不符；Ｂ２２亚型以３＾＊５４０１最多，Ｂ＾＊５６０１较少，１例分型格局异常，可能为新Ｂ２２等位基因或错配的     

HLA—DR位点的PCR—SSP基因分型

目的：探讨适用于肾移植供体HLA-DR的快速基因分型方法。方法：自行设计合成引物建立顺序特异性引物聚合酶链反应技术,并对110例肾移植供体作HLA-DR基因分型。结果：DNA提取方法可以满足PCR-SSP分型方法对DNA模板的要求。全部操作耗时120min,110例标本均分型成功。基因频率范畴在0.0045-0.1227之间,以DR4、DR15和DR9占多数。结论：PCR-SSP法作HLA-DR分型具有准确,简便,快速等优点。适合在临床进行推广应用。     

HLA-DR位点的基因芯片分型与临床应用

目的：建立一种新型的HLA－DR位点的基因分型方法，为HLA－DR的基因分型提供一个较新的思路。方法：利用基因芯片技术HLA不同基因亚型的独特序列设计探讨，制成分型芯片；待检测样品经PCR反应标记上荧光之后，与芯片进行杂交，根据杂交产生的荧光信号值分析确定样品DR位点的基因亚型，将这一方法应用于70份标准DNA和200份医院移植供受者的HLA－DR基因分型并将部分样品进行基因测序。结果：检测结果表明HLA－DR基因分型芯片可准确分辨出DR位点等位基因30大类，耗时3h 。结论：基因芯片用HLA－DR的基因分型，分辨率高，特异性强，重复性好，操作简便，对比常规的PCR－SSP和PCR－SSO方法，HLA－DR基因芯片方法更为直观，并具有集成化优势。可以在一张芯片上同时检测HLAⅠ类的A、B位点，并实现一张芯片多人份，适合于临床应用和骨髓库，脐血库的建立。     

采用 PCR-SSP法检测HLA-B27基因

目的：建立顺序特异引物聚合酶链反应（ＰＣＲ－ＳＳＰ）方法检测ＨＩＡ－Ｂ２７基因并与血清学方法比较。方法：设计合成Ｂ２７特异物３个和内源性阳性对照２个,建立ＰＣＲ－ＳＳＰ方法,用于Ｂ２７基因分型。血清学分型为一步法单克隆抗估方法。临床样本１００份,不源于可疑强直性脊柱炎患者。快速酚氯仿法提取模板ＤＮＡ。结果：１００例临床样本ＰＣＲ－ＳＳＰ行Ｂ２７基因分型均获成功。总耗时４ｈ。其中Ｂ２７阴性５８例,阳     

HLA-B40组等位基因多态性和血清学分型不明确标本分析

目的：利用DNA分型技术调查上海汉族人群HLA－B40组等位基因多态性并比较配型标本中HLA－B40抗原血清学和DNA分型的结果：方法：采用反向PCR－SSOP技术进行DNA分型，可检出HLA－B＊4001－4011等11个等位基因，结果：所有标本DNA分型均获成功，无假阳性和假阴性结果出现，质控DNA分型结果与UCLA结果相符，上海地区汉族人群共检出B＊4001－4003，4005－4007，4011等等位基因，未检出B＊4004，4008－4010等等位基因，296名无关个体中HLA－B40＊组等位基因频率为0．1402，血清学方法检测HLA－B40组抗原错误率为12．82％（10／78），结论：该技术用于HLA－B40分型分辨率高，分型结果较血清学方法更加精确，可确保HLA分型的准确。     

116例脐血PCR-SSP和PCR-SSOP的HLA-DR分型结果分析

脐血中富含造血干细胞。造血干细胞移植是治疗多种危重病患者的有效手段。移植前的ＨＬＡ分型是决定其移植成功的重要环节之一。器官移植供受者间ＨＬＡ ＤＲ不相容可引起急性移植物抗宿主反应 (ＧＶＨＤ)。因此 ,脐血移植中需有精确的ＨＬＡ ＤＲ基因分型。本研究从 1999年 1月至 7月对脐血库的 116例标本分别用ＰＣＲ ＳＳＰ和ＰＣＲ ＳＳＯＰ法检测 ,分析结果如下。材料和方法标本来源 :116例标本来自本院脐血库中的脐血标本。标本的ＤＮＡ提取 :脐血标本为ＡＣＤ Ｂ抗凝血 ,取 2 0 0 μｌ作盐酸胍法抽提ＤＮＡ〔1〕。ＰＣＲ ＳＳＰ方法 …     

HLA-B27基因在强直性脊椎炎中的临床应用

目的：为评价ＨＬＡ－Ｂ２７基因在强直性脊椎炎的临床诊断价值。方法：采用ＰＣＲ－ＳＳＰ地１４１例风湿性病人ＨＬＡ－Ｂ２７工与血沉、影像学Ｘ光片报告开面指标进行比较。结果：强直性脊椎炎病人组ＨＬＡ－Ｂ２７是性率６８．８％与风湿必 人组ＨＬＡ－Ｂ２７基因阳性率１１．４％,比较,Ｐ〈０．００５,有非常显著性划;强直性脊椎炎病人Ｂ２７基因阳性率６８．８％,与血沉结果升高率８７．５％比较,Ｐ〉０．０５,无显著     

HLAⅡ类基因PCR-SSP分型技术在骨髓移植配型中的应用

建立快速、准确的ＨＬＡ基因分型方法,满足临床移植配型的需要。方法通过序列特异性引物聚合酶链反应,对拟行骨髓移植的１０例血液病患者及１２名相关供者的ＨＬＡ－Ⅱ类基因ＤＲＢ１,ＤＱＢ１位点扩增,琼脂糖凝胶电泳分析ＰＣＲ产物并确定其基因型。结论该方法具有快速准确,特异性高,简便省时的特点。     

HLA-B27 testing in Thai patients using the PCR-SSP technique

We develop the HLA-B27 test kit using the PCR-SSP technique. Five hundred forty blood samples were tested for HLA-B27 by microlymphocytotoxicity test (LCT) and PCR-SSP. It was found that 127 (23.5%) and 134 (24.8%) of these samples were positive for HLA-B27 by LCT and PCR-SSP, respectively. The sensitivity and specificity of the PCR-SSP were 94.8 and 100%, respectively, when using LCT as the standard method. The PCR-SSP positive predictive value was 100%, negative predictive value was 98.3%, and a concordance rate of 98.7%. This study shows that the PCR-SSP is simple, convenient, and a more cost-effective in-house test kit.     

Allele-specific HLA-B*15 typing by PCR-SSP and its application to four distinct ethnic populations

Abstract: We present a set of primer mixes for the allele-specific typing of the HLA-B*15 group by PCR-SSP. The set comprises 46 primer mixes which are designed to unequivocally resolve all but two of the 666 possible combinations of the B*15 alleles, B*1501–37 (B*1536 sequence unavailable). A core subset of 34 of the 46 mixes can be used alone to give a high resolution B*15 typing set. This allows for the identification of each B*15 allele when present as the only B*15 allele and the majority of the possible B*15 homozygotic combinations. The method was validated using reference DNA samples and the B*15 allele frequency in 4 distinct ethnic populations was investigated. The results show that these populations contain predominantly mutually exclusive sets of B*15 alleles.     

用PCR-SSP作HLA-B位点分型及B22亚型分析

目的:用DNA分型弥补血清学HLAB位点分型的欠缺并了解南方汉族B22亚型分布情况。方法:采用标准细胞作参照,建立B位点的PCRSSP分型方法;并对血清学B22阳性标本进行B22亚型分析。结果:104株标准细胞PCRSSP分型结果与已知结果完全一致,17例白血病人及同胞分型与血清学一致,1例不符;B22亚型以B5401最多,B5601较少,1例分型格局异常,可能为新B22等位基因或错配的DNA双链。结论:PCRSSP可用于B位点的DNA分型,比血清学更直观精确,操作简便,不受疾病状态影响。     

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP)

Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.     

Identification of a new HLA-B*39 allele: HLA-B*3924

We have identified a new HLA-B*39 allele through polymerase chain reaction (PCR) using sequence-specific primers (SSP) and sequence-based typing of exons 2 and 3. This novel allele was identified in three HLA-identical siblings of Turkish origin. This allele only differs from HLA-B*3903 at a unique single nucleotide substitution (T for C) at position 365 in exon 3 which results in an amino acid change in codon 98 of methionine (ATG) to threonine (ACG). The sequencing enabled the development of a monospecific PCR-SSP reaction which can be used to discriminate between HLA-B*3924 and other B*39 alleles.     

Development of a multiplex PCR-SSP method for Killer-cell immunoglobulin-like receptor genotyping

Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells recognize groups of HLA class I alleles. Recent work suggests that KIR genotype may affect the outcome of hematopoietic stem-cell transplants and that prospective KIR typing maybe of benefit in future matching of donors and recipients. A simple and informative KIR genotyping method was developed using a multiplex polymerase chain reaction-sequence-specific primer strategy. This method contains four multiplex reactions for detecting all functional KIR genes, including some 2DS4 variants that harbor a common deletion. Primer pairs were designed to provide short amplicons (108-565 bp) that can be analyzed by agarose gel electrophoreses or by automated electrophoretic systems. This method was evaluated in a blinded survey with the NK/KIR Phase II QC Panel (a total of 16 cell lines) from the 14th International Histocompatibility Workshop (IHWS), and the results are 100% concordant with the consensus genotype. Results in further KIR genotyping of 20 reference cell lines from the 10th IHWS were consistent with previously published genotypes, matching those of one study in instances where different genotypes have been previously reported. The genotypes obtained in this study may be helpful to other labs developing KIR genotyping methods in resolving typing discrepancies and in detecting common deletion variants of 2DS4. This method can save labor and reagent costs. It provides good results from partially degraded template DNA due to short amplicons in this method. It is convenient to use in both clinical and research laboratories.     

Identification of a new HLA-B*15 allele,HLA-B*1569

We have identified a new HLA-B*15 allele (B*1569) by polymerase chain reaction (PCR) using sequence-specific primers (SSP) and sequence-based typing (SBT). This novel allele was found in a 67-year-old white Caucasian male and differs from HLA-B*1503 at 3 positions. The nucleotide substitutions at positions 544, 559 and 560 result in amino acid changes at codon 158 from GCC (alanine) to ACC (threonine), and at codon 163 from CTG (leucine) to ACG (threonine).     

Low resolution DNA typing of the HLA-B5 cross-reactive group by nested PCR-SSP

Abstract: We have established a DNA typing system for the HLA-B5 serologically cross-reactive group (CREG) by means of a two-step PCR amplification with nested sequence-specific primers (nPCR-SSP). The present study provides a low resolution definition of the HLA-B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group-specific amplification of all HLA-B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA-B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n=50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA-B specificities by nPCR-SSP.